BackgroundPrednisolone is considered the cornerstone treatment for severe alcoholic hepatitis (AH). However, its use is limited by the increased risk of infection in an already immunocompromised patient population. Among patients with severe AH, there exists a group of non-responders who do not benefit from prednisolone therapy. Day-4 Lille score is a widely employed prognostic model used to identify this non-responder subgroup. The present study evaluates the prognostic ability of the inflammatory marker, the neutrophil-lymphocyte ratio (NLR), as a stand-alone model and in conjunction with the day-4 Lille score.MethodsWe retrospectively reviewed the electronic medical records of patients diagnosed with AH. Demographic and biochemical data at diagnosis were collected to calculate Maddrey’s discriminant function (MDF) and model for end-stage liver disease (MELD) score upon admission and also on day 4. Receiver operating characteristic (ROC) curves were plotted for day-4 NLR and day-4 Lille score for prediction of 90-day mortality, and optimal cut-off values were determined. Patients were then subcategorized into groups based on the generated optimal cut-off values. Categorization was validated by comparing the mortality rate in each group with the chi-squared test. We then performed a multivariate analysis for prediction of 90-day mortality using day-4 Lille score and day-4 NLR, constructing a new prediction score based on the odds ratio (OR). The ROC curve of the new score was plotted and the area under a curve (AUC) was reported and compared with previously validated scores.ResultsOur analysis demonstrated that both day-4 NLR and Lille score individually predicted 90-day mortality with statistical significance . The ROC analysis of day-4 Lille score for the prediction of 90-day mortality revealed an AUC of 0.819 with an optimal cut-off value of 0.45 . Day-4 NLR had an AUC of 0.756 with an optimal cut-off value of 12.3  The combined day-Lille-NLR model with a cut-off of 0.55 had an AUC of .889, which was higher than day-4 Lille score and NLR independently.ConclusionDay-4 NLR is an easily assessed prognostic model of mortality in alcoholic hepatitis. However, it often underperforms relative to day-4 Lille score. Combining these two models to create a "modified" Lille score adds increased performance characteristics to the prediction of outcomes/mortality. The "modified" Lille score presented in this study can be used to further cut down the number of non-responders who are often forced to undergo costly and potentially harmful treatment courses. Alcohol abuse is one of the leading causes of preventable disease-associated mortality globally . The priAH is an acute inflammatory condition characterized by hepatocellular injury mediated by alcohol-induced oxidative stress . Mild anOnce an astute clinician identifies AH in a patient, management relies on initial prognostication. There are multiple scoring systems for risk-stratification; Maddrey discriminant function (MDF); model for end-stage liver disease (MELD) score; Glasgow alcoholic hepatitis score (GAHS); age, serum bilirubin, INR, and serum creatinine (ABIC) score; and Lille score. Among these, MDF is commonly used to predict 28-day mortality and identify severe AH, which would benefit from the primary therapeutic interventions: prednisolone and pentoxifylline. MDF is commonly used in conjunction with Lille scores to identify a subgroup of non-responders. Cessation of corticosteroids early in the course of treatment in non-responders is crucial to avoid complications of treatment such as infection and acute kidney injury.Currently, there is an evolving body of literature regarding the concept of the neutrophil-lymphocyte ratio (NLR) and its correlation with the severity and prognosis in a variety of gastrointestinal inflammatory diseases including acute appendicitis, acute pancreatitis, and hepatitis B -9. In thDatabaseWe retrospectively reviewed the electronic medical records of patients who were diagnosed with AH during hospitalization at John H. Stroger, Jr. Hospital in Cook County, Chicago, IL, from January 1, 2010 to July 31, 2016. We identified potential patients using the ICD-9 and ICD-10 discharge diagnosis codes. We then reviewed the demographic, clinical, and laboratory data to confirm the diagnosis of AH based on the following criteria: (1) ongoing alcohol consumption for six months or more, exceeding 60 g/d for male and 40 g/d for female; (2) aspartate aminotransferase level of >50, aspartate aminotransferase/alanine aminotransferase level >1.5-fold than normal, and both values <400 IU/L; (3) no known differential diagnoses of acute hepatitis including: acetaminophen toxicity, acute viral hepatitis, ischemic hepatitis, Budd-Chiari syndrome, autoimmune hepatitis, drug-induced liver injury [The present study was approved by the Institutional Review Board of the Cook County Health & Hospitals System, Chicago. IL. The database was set up and maintained by the Department of Medicine, Cook County Health & Hospitals System.VariablesDemographic variables including age, gender, medical history, and alcohol, tobacco, and illicit substance use were abstracted. We obtained biochemical data at diagnosis to calculate MDF and MELD score upon admission. We then extracted biochemical data at day 4 of hospitalization to calculate the day-4 Lille score, MELD score, and NLR. Prognosis at 90 days was confirmed in all patients during in-person or phone follow-ups.Statistical analysisWe performed analyses to describe and summarize the distributions of variables. Receiver operating characteristic (ROC) curves were plotted for day-4 NLR and day-4 Lille score for prediction of 90-day mortality, and optimal cut-off values were determined. Patients were then subcategorized into groups based on the optimal cut-off values, and mortality rate in each group compared with the chi-square test to further validate the categorization. Finally, we performed multivariate analysis for the prediction of 90-day mortality using the day-4 Lille score and day-4 NLR and constructed a new prediction score based on the odds ratio (OR). The ROC curve of the new score was plotted and the area under the curve (AUC) was reported in comparison with pre-existing validated scores. All statistical analyses were performed using STATA Version 14.0 . We considered p-values of <0.05 to be statistically significant.Cohort characteristicsWe identified 104 patients with a confirmed diagnosis of severe AH requiring prednisolone therapy  group had 20.5% mortality, significantly higher than that of the low-NLR group . High-Lille-score (>0.45) group had 31.2% mortality, significantly higher than that of the low-Lille-score group  . The significance for both scores remained in multivariate analysis Table . We consAH is a disease resulting from excessive alcohol consumption and is characterized by marked inflammation both within the liver parenchyma and systemically. The pathogenesis of AH is mediated by two mechanisms: (1) lipopolysaccharide/endotoxin entry through a leaky small intestine and subsequent activation of the innate immune system; and (2) direct activation of the complement pathway by alcohol. The outcome of either pathway is the production of chemoattractants and the recruitment of neutrophils. Elevated neutrophils in the blood, even in the absence of bacterial or fungal infections, is a condition not found in other ailments of the liver . SeveralA more cost-effective and accessible biomarker of the inflammatory state, NLR, has not yet been validated in the AH treatment paradigm. Nevertheless, NLR has demonstrated efficacy as a prognostic tool in a variety of inflammatory conditions. In many GI malignancies, including hepatocellular, colorectal, esophageal, and pancreatic, NLR has the ability to predict outcomes post-surgery and after treatment -14. AddiPrior to prognostication with either Lille or NLR, patients are risk-stratified into mild, moderate, or severe AH categories based on MDF. Patients with severe AH are treated with prednisolone or, when contraindications to prednisolone are present, with pentoxifylline. After therapy for 4 to 7 days, the Lille score is calculated to assess the response to therapy and recognize non-responders. Using age, albumin, bilirubin, creatinine, and prothrombin time, the Lille model can prognosticate AH with readily available laboratory studies. A Lille score >0.45 correlates with poor outcomes despite prednisolone and represents non-response to therapy . AccordiOur investigation found that day-4 NLR has significant prognostic value. With a cut-off value of 12.3, NLR can help categorize patients with severe AH into high-mortality (20.5%) and low-mortality (5.0%) groups. Compared to the day-4 Lille score, NLR with a cut-off value of 12.3 has comparable specificity (78.1% in NLR vs 76.1% in Lille) but worse sensitivity (66.7% in NLR vs 83.3% in Lille). As an isolated prognostic model, NLR’s value relies on its simplicity and ease of use in patients lacking complete lab results on day 4 to calculate a Lille score. In most patients, this does not represent a significant issue, and thus we sought to evaluate the efficacy of Lille and NLR predictive models applied synchronously. As the Lille score does not account for white blood cell count or its derivatives, we created a “modified” Lille Score that directly merged day-4 NLR and day-4 Lille. In the area under ROC analysis, this “modified” Lille score outperforms either Lille or NLR models individually. With an optimal cut-off of 0.55, the “modified” Lille Score leads to a more recognizable delineation of non-responders and subsequently results in improved management of severe AH in two ways: (1) by reducing exposure to non-beneficial glucocorticoids; and (2) by allowing for the study of alternative therapies in the non-responder group.Day-4 NLR is an easily calculable model for the prediction of mortality in AH. However, though day-4 NLR can predict mortality in a statistically significant manner, it usually underperforms compared to the Lille score. A “modified” Lille score that incorporates both NLR and Lille prognostic models was assessed and demonstrated to have better performance in prediction of mortality than each individual model. Our “modified” Lille score allows for improved identification of patients benefiting from the early termination of corticosteroids and thus reduces iatrogenic complications of prolonged steroid therapy.


Diploptera functata (D. functata) has a higher nutritional potential than conventional mammalian milk. It contains rich stores of essential nutrients such as oleic acid, conjugated linoleic acid, omega-3 fatty acids, short-chain and medium-chain fatty acids, vitamins and minerals . FurtheD. functata is a cockroach species known to give birth to live offspring. It has the ability to produce milk in the form of a substance containing protein crystals that serves as nutrition for its young. This so-called 'cockroach milk' is a source of N-acetyl-D-glucosamine, β-D-mannose, oleic acid, linoleic acid and glycerol  the energy content of milk produced by buffalo's and other mammals as shown in Figure 1D. functata milk protein crystals are highly heterogeneous , has a protein content far superior to commercial flour acquired from wheat (63.22 % versus 9.8 %). This 'insect flour' contains eight essential amino acids and a high quantity of omega-3 (ω-3) and omega-9 (ω-9) fatty acids. Amino acid analysis revealed that flour produced from the N. cinerea cockroach contains leucine, lysine, and valine at percentages of 3.51, 3.37 and 2.61 % respectively. As for fatty acids, the flour consists of 5.69 % palmatic acid, 9.17 % oleic acid, 7.51 % saturated- and 10.94 % unsaturated fatty acids, all of them acknowledged components of a healthy diet (De Oliveira et al., 2017[N. cinerea flour's benefits could be effectuated without any loss of gustatory quality. Superfoods like the aforementioned cockroach milk and cockroach flour are expected to play a pivotal role in the solution to food shortage in the decades to come (De Oliveira et al., 2017[A recent study conducted in Brazil revealed that flour made of another breed of cockroaches, Milk protein crystals and other products produced from cockroaches could be part of a next generation of super foods. Milk is a valued component of the human diet as its nutritional properties enhance human health and quality of life. It is known that milk contributes to the prevention of certain diseases and reduces certain threats to health. Several authors mention milk in association with a reduction in risk of cancer, ischaemic heart disease and diabetes (Elfahri et al., 2016; Hove etThere is no conflict of interest.The authors who worked on this manuscript acknowledge their respective universities and institutes.


Staphylococcus aureus, Escherichia coli and Bacillus subtilis. The Janus nanoparticles had larger minimal inhibitory concentration (MIC) and minimum bacterial concentration (MBC) values than the multi-core shell counterparts. The results of cytotoxicity showed the Janus Ag-PEG nanoparticles had lower toxicity than the multi-core shell nanoparticles.Silver nanoparticles synthesized with polymers as coating agents is an effective method to overcome their poor stability and aggregation in solution. Silver-polyethylene glycol (Ag-PEG) nanoparticles were synthesized with the thiol-functionalized polyethylene glycol (SH-PEA) as the coating, reducing and stabilizing agent. The UV irradiation time, polymer and silver nitrate concentration for the synthesis were investigated. The concentration of silver nitrate had significant effect on the morphology of Ag-PEG nanoparticles. When increasing the concentration of silver nitrate, SEM and TEM images showed that Ag-PEG nanoparticles changed from Janus to multi-core shell structure. Meanwhile, pure silver particles in the two hybrid nanoparticles presented spherical shape and had the similar size of 15 nm. The antibacterial activities and cytotoxicity of the two structural Ag-PEG nanoparticles were investigated to understand colloid morphology effect on the properties of AgNPs. The results of antibacterial activities showed that the two structural Ag-PEG nanoparticles exhibited strong antibacterial activities against  Silver nanoparticles (AgNPs) have attracted increasing interest due to their unique physical, chemical and biological properties, including surface-enhanced Raman scattering, catalytic activity, non-linear optical behavior and broad-spectrum bactericidal activity ,6,7,8,9.Janus particles possess two or more different chemical components and spatial distribution that have different surface, structures or material properties ,24. AlthIn this paper, we present a facile and fast approach for the fabrication of silver-polyethylene glycol (Ag-PEG) nanoparticles with tunable morphologies in Janus and multi-core shell structure which was performed by photochemical reduction method with thiol-functionalized polyethylene glycol (SH-PEA) as the coating, reducing and stabilizing agent. Optimization of different experimental variables like UV irradiation time, polymer concentration and silver precursor concentration were systematically investigated for the two structures of Ag-PEG nanoparticles. Silver colloids are usually stable against auto-aggregation which is potential materials in antibacterial and anticancer medicine. Thus, the biological properties of Ag-PEG nanoparticles had been concerned about. To understand the effect of the morphology of the hybrid nanoparticles on their biological properties, the antibacterial activities and cytotoxicity of the Janus and multi-core shell Ag-PEG nanoparticles were assessed by bacterial growth inhibitory and 3--2,5-dephenyl tetrazolium bromide (MTT) cell viability assays, respectively.Mn = 500 g·mol−1) was obtained from Sigma-Aldrich Co. . Silver nitrate (AgNO3), alcohol and n-hexane were purchased from Tian Jin Chemical Reagent . 3--2,5-ziphenyl tetrazolium bromide (MTT) was purchased from Amresco Inc. . Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL Co. . The phosphate buffer saline (PBS) was made in laboratory according to the literature [Staphylococcus aureus (ATCC 6358P), Escherichia coli (CGMCC 1.90) and Bacillus subtilis (CGMCC 1.1849) were used for the bacterial studies. All microorganisms were provided by China General Microbiological Culture Collection Center (CGMCC), Chinese Academy of Sciences (CAS), Beijing, China. The bacterial strains were grown in Luria-Bertani agar plates from Hopebiol  at 37 °C.Mercapto-ethylamine (MEA) was purchased from Xiya Reagent . Poly(ethylene glycol) diglycidyl ether  spectra were collected on an AVANCE III HD 400 MHz spectrometer  in CDCl3 at 25 °C. Gel permeation chromatography (GPC) measurements were conducted with a Waters 410 GPC  equipped with Waters Styragel column (HT4 + HT3) using CDCl3 as the eluent, the molecular weights were calibrated with polystyrene standards, and the flow rate was set at 1.0 mL min−1 at 35 °C. UV-Vis absorption spectra were recorded by using a U4100 spectrophotometer  with the as-prepared silver colloidal solution. The surface morphology of samples was analyzed on a JSM-7610F  scanning electron microscope (SEM) and the main elements were measured by energy-dispersive X-ray spectrometry (EDS) . The samples on the silicon wafers were mounted rigidly to a copper specimen holder using a conductive adhesive. Transmission electron microscopy (TEM) studies were performed on a JEM-2100 electron microscope  operating at an acceleration voltage of 100 KV. The nanoparticle solution was dropped on copper grids, and dried at room temperature. X-ray diffraction (XRD) was recorded on a D 8 Advance . The samples were drop cast on glass slides, and dried at room temperature.FT-IR spectra were recorded in a FT-IR SPECRUM VERTEX 70 spectrometer  on thoroughly dried samples by using KBr pellets. SH-PEA was synthesized following the procedure conducted by Wen and coworkers . A mixtu3 (4 mg) were dissolved into 20 mL distilled water at room temperature. After 3 h, the mixture solution was subjected to UV-irradiation with a UV light  at an intensity of 75 mW·cm−2 at 365 nm for 3 min. The reaction mixture was extensively dialyzed against water for 3 days to remove the excess reactants. The optimization of different experimental variables of UV irradiation time, SH-PEA concentration and silver nitrate concentration were investigated systematically.The synthesis of Ag-PEG nanoparticles was carried out in a typical experiment. SH-PEA (20 mg) and AgNOE. coli, S. aureus and B. subtilis according to the previous report [6 CFU mL−1) was inoculated evenly on a Luria-Bertani agar plate. Then, the Oxford cups were placed on the surfaces of the cultures, and an amount of 200 μL solution of the nanoparticle (1 mg mL−1) was place into the oxford cups. After incubation for 24 h at 37 °C, the antibacterial activity was evaluated by measuring the average diameters of the zones of inhibition with a vernier calliper for three times.Oxford cup method was used to determine the antibacterial activity with different bacterial such as s report . The bac−1). Distinct volumes of the stock solutions were diluted by 10 mL growth medium to form series of solutions with different concentration of Ag-PEG nanoparticles. Then, these solutions were inoculated with 100 μL of a bacterial suspension (105 CFU mL−1) and then cultured for 1 day in a 37 °C incubator and immediately used in MIC assays. To determine MIC, the cultures were analyzed for the absence of turbidity. A total of 3 replicates were carried out for each treatment and the corresponding controls.The minimal inhibitory concentration (MIC) was read by the visual turbidity of the tubes noted before and after incubation according to previous reports ,34. The The minimum bacterial concentration (MBC) was determined according to the previous report . After t4 cells per well and incubated in DMEM (100 mL) for 24 h. Then, seven concentrations  of Ag-PEG were added to the wells. Three parallel wells for each sample were used at a specific concentration. After co-incubation with cells for 48 h, 20 μL of MTT solution in PBS (5 mg mL-1) was added to each well and the plate was incubated for another 4 h at 37 °C. After that, the medium containing MTT was removed, and 150 μL of DMSO was added to each well to dissolve the MTT formazan crystals. Finally, the plates were shaken for 5 min, and the absorbance of formazan product was measured at 490 nm by a Bio-Rad 680 micro-plate reader .The cytotoxicity of Ag-PEG was examined by MTT assay. All sample solutions were diluted with DMEM to obtain preset concentrations. HepG2 and MCF-7 cells were seeded into 96-well plates with a density of 10PEG is a hydrophilic polymer with excellent biocompatibility and widely used to modify metal or metal oxide nanomaterials for biomedicine application ,37,38, w1H NMR spectra were used to study chemical structure of SH-PEA. SH-PEA has one characteristic absorption peak at 3396 cm−1, attributed to the stretching vibration of the -OH groups. The peaks at 2875 cm−1 belongs to the stretching vibration of C-H bond . The data shows the Mn and polydispersity for SH-PEA is 0.67 × 104 g mol−1 and 2.41.FT-IR and C-H bond a. The as3 with SH-PEA in water.The functionality of SH-PEA makes it possible to utilizing photochemical reduction route for the fabrication of AgNPs with a green and facile method. Photochemical reduction has been proved as a cost-effective, convenient and controllable technique to prepare AgNPs ,42,43. I−1, corresponding to the stretching peaks of tert-amine, shifts to 1638 cm−1 with a significant decrease in the transmittance. Meanwhile, the intensity of the C-S stretching band around 656 cm−1 almost disappears in the spectrum of Ag-PEG nanoparticles.The formation of Ag-PEG nanoparticles was confirmed by FT-IR spectroscopy. As shown in −1 AgNO3 and 1 mg mL−1 SH-PEA at different time. It is obvious that no AgNPs formed without UV irradiation, even though the aqueous solution of SH-PEA and AgNO3 was stirred in dark for 3 h. However, the color of the solution changed from colorless to brown when the solution was irradiated for several minutes, indicating the reduction of silver ion to silver and the formation of AgNPs. An absorption band was existed at 414 nm after irradiation for 1 min, and the absorbance increases with increasing irradiation time. A single symmetric band with one maximum around 400 nm for the surface Plasmon resonance are attributed to the typical absorption spectrum of spherical AgNPs [The optical properties of Ag-PEG nanoparticles were investigated using UV-Vis spectrometry. al AgNPs . Althougθ = 38.4° and 43.2° index to (111) and (200) planes of Ag.The morphologies and corresponding silver domain size distributions of Ag-PEG nanoparticles observed by using SEM and TEM are shown in −1 AgNO3, and monitored their surface Plasmon resonance with a UV-Vis spectrometer. In −1. Furthermore, increasing SH-PEA concentration, the band turns broader and takes a red shift to 428 nm, which indicated that the AgNPs grow up or aggregate. −1 SH-PEA. Large Ag-PEG nanoparticles and agglomeration are observed, and the size of pure AgNPs is over 50 nm, much larger than the size of Ag-PEG nanoparticles synthesized with 0.2 mg mL−1 SH-PEA. Besides, both Ag and PEG domains become irregular although the nanoparticles remain Janus structure. It is obvious that the lower SH-PEA concentration is appropriate for the stable Ag-PEG nanoparticles.To investigate the influence of SH-PEA concentration on the size of AgNPs, we synthesized Ag-PEG nanoparticles with different SH-PEA concentration and 0.2 mg mL3 used as the precursor is an important variable in the synthesis of AgNPs. However, few researches have studied the effect of silver ion on the size, shape or properties of AgNPs. In this synthesis process, we found that the concentration of AgNO3 played a key role in the morphology of Ag-PEG nanoparticles. 3 concentration from 0.2 to 1 mg mL−1. −1 AgNO3, respectively. They are very similar as the result of Ag-PEG nanoparticles with 0.2 mg mL−1 AgNO3 shown in 3 concentration is 0.5 mg mL−1, a multi-core shell structure nanoparticles with the size about 100 nm exists in the image as shown in 3 concentration is up to 1.0 mg mL−1, the Janus nanoparticles almost disappear, and they are replaced by the multi-core shell nanoparticles  values of the Janus nanoparticles were much higher than that of the multi-core shell nanoparticles. The MBC values of Janus Ag-PEG nanoparticles were 500 μg mL−1 against S. aureus and E. coli, and 250 μg mL−1 against B. subtilis, while the values of multi-core shell nanoparticles were 64 μg mL−1 against S. aureus and 32 μg mL−1 against E. coli and B. subtilis. The MBC/MIC ratio shows the two structural nanoparticles have different antibacterial properties. The ratio of the Janus Ag-PEG nanoparticles is higher than 4, suggesting that the Janus Ag-PEG can be considered as a bacteriostatic agent. On the contrary, the multi-core shell Ag-PEG nanoparticles have lower MBC/MIC ratio (≤4), performing with bactericidal manner [A large decrease in the optical absorbance of the bacterial suspensions was observed when they were exposed to Ag-PEG nanoparticles. As shown in l manner .−1). The percentage of viable cells is always higher than 80%. The Janus Ag-PEG nanoparticles tested in different concentrations in the range of 4–250 μg mL−1 shows a high percentage (around 90%) of survival cells as its concentration is below 64 μg mL−1. Subsequently, a significant drop happens in their number for higher concentrations, and the drop of MCF-7 does faster. Instead, the multi-core shell Ag-PEG nanoparticles showed very high toxicity to HepG2 and MCF-7 cells. The percentage of viable cells for the multi-core shell nanoparticles against HepG2 is lower than 10% when its concentration is excess over 8 μg mL−1. To MCF-7, the number is only 81% even though the concentration of the nanoparticles is as low as 4 μg mL−1. The result demonstrates that the Janus Ag-PEG nanoparticles are more biocompatible than the multi-core shell with the investigated cells.MTT assay was used to evaluate the cytotoxicity of Ag-PEG nanoparticles by using HepG2 and MCF-7 cell lines, and the results were shown in −1 SH-PEA is optimized for synthesizing stable Ag-PEG nanoparticles. The AgNO3 concentration plays a key role in tuning the morphologies of Ag-PEG nanoparticles. Janus Ag-PEG nanoparticles were synthesized with lower AgNO3 concentration, and multi-core shell structure with higher AgNO3 concentration. The pure AgNPs show spherical shape and have the size about 15 nm in the two structural Ag-PEG nanoparticles. Both structural Ag-PEG nanoparticles present strong antibacterial activities against E. coli, S. aureus and B. subtilis. The MIC and MBC values of the Janus nanoparticles are larger than those of the multi-core shell ones. The MBC/MIC ratio of the former is higher than 4, while the latter is lower than 4. HepG2 and MCF-7 cells were used to evaluate the cytotoxicity of the Ag-PEG nanoparticles. The Janus Ag-PEG nanoparticles show good biocompatibility when its concentration is below 64 μg mL−1, while the multi-core shell nanoparticles perform high toxicity even its concentration is as low as 8 μg mL−1. The results demonstrate the morphology of Ag-PEG hybrid nanoparticle has important effect on its biological properties.In summary, we synthesized Ag-PEG hybrid nanoparticles by photochemical reduction method with the as-synthesized SH-PEA as the coating, reducing and stabilizing agent. The condition of 3 min irradiation and 1 mg mL


N-isopropylacrylamide (NIPAAm), and EGMA were investigated. The molar ratios of NIPAAm and EGMA were varied from 20:0 to 19.5:0.5 and 18.5:1.5 in the thermo-responsive alginate-Ca2+/P(NIPAAm-co-EGMA) IPN hydrogels. Due to the more hydrophilicity and high flexibility of EGMA, the IPN hydrogels exhibited higher lower critical solution temperature (LCST) and lower glass transition temperature (Tg) when the ratio of EGMA increases. The swelling/deswelling kinetics of the IPN hydrogels could be controlled by adjusting the NIPAAm/EGMA molar ratio. A faster water uptake rate and a slower water loss rate could be realized by increase the amount of EGMA in the IPN hydrogel . By using 2-Isopropylthioxanthone (ITX) as a photo initiator, the obtained alginate-Ca2+/P(NIPAAm-co-EGMA360) IPN hydrogels were successfully immobilized on cotton fabrics. The surface and cross section of the hydrogel were probed by scanning electron microscopy (SEM). They all exhibited a porous structure, and the pore size was increased with the amount of EGMA. Moreover, the LCST values of the fabric-grafted hydrogels were close to those of the pure IPN hydrogels. Their thermal sensitivity remained unchanged. The cotton fabrics grafted with hydrogel turned out to be much softer with the continuous increase of EGMA amount. Therefore, compared with alginate-Ca2+/PNIPAAm hydrogel, alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogel is a more promising candidate for wound dressing in the field of biomedical textile.The influence of ethylene glycol methacrylate (EGMA) to the hydration and transition behaviors of thermo-responsive interpenetrating polymeric network (IPN) hydrogels containing sodium alginate,  N-isopropylacrylamide) (PNIPAAm) gel is a typical thermosensitive hydrogel that exhibits an abrupt volume transition in response to change of temperature around 33 °C. Its characteristic lower critical solution temperature (LCST) is close to human body temperature [Hydrogels are three-dimensional polymeric networks with hydrophilic groups that can absorb from 10–20% up to hundreds of times their dry weight in water ,4, pH 55, solvenperature ,10. LCSTperature , tissue perature , cell atperature , proteinperature , etc. Hoperature .2+ and poly(N-isopropylacrylamide) and investigated the temperature- and pH-modulated equilibrium swelling and drug release with indomethacin. Zhang G. et al. [Tg) [Sodium alginate (SA) is a well-known natural polysaccharide extracted from brown seaweed, which is a promising candidate to obtain hydrogels due to its properties of abundant, biocompatibility, biodegradability, renewable, non-toxic and a relatively low cost . Combini. et al.  presente. et al.  presenteal. [Tg) , which wTg (blow 0 °C); moreover, the LCST of poly(methacrylate)s can be easily adjusted between 5–90 °C by changing the number of ethoxy groups in the side chains [2+/PNIPAAm IPN hydrogel, the alginate-Ca2+/P(NIPAAm-co-EGMA) IPN hydrogels obtained presented the reduced Tg and provided additional hydroxyl groups to enhance the swelling capability. Hence, the modified hydrogels will be suitable for the applications in the field of textiles, such as the wound dressing.Besides the acrylamide hydrogels, another class of thermo-responsive polymers based on poly(methacrylate)s has arose the interests of researchers ,25,26. Ce chains . Hence, 2+/P(NIPAAm-co-EGMA360) was synthesized via free-radical polymerization. Thermogravimetry analysis (TGA), differential scanning calorimetry (DSC), and swelling/deswelling ratio were applied to characterize the thermal behaviors of IPN hydrogels, and in order to compare these thermal behaviors, IPN hydrogels were prepared by different NIPAAm/EGMA ratios. Afterward, they were grafted with the cotton fabric by UV photo-grafting. The surface and cross section morphology, temperature-dependent swelling behavior and hand evaluation were discussed to evaluate the cotton fabric grafted hydrogels. Finally, a conclusion was given to summarize the variation of the thermal behaviors of the IPN hydrogels and the hand feeling of cotton-fabric composites.In this paper, the thermo-responsive hydrogel alginate-Caw/v water solution at 25 °C), and ethylene glycol methacrylate  (EGMA360) was purchased from Sigma-Aldrich Co., Ltd. . Ammonium persulfate (APS), N,N′-Methylenebisacrylamide (MBAA), N-isopropylacrylamide (NIPAAm), calcium chloride (CaCl2), N,N,N′,N′-Tetramethylethylenediamine (TEMED) and 2-Isopropylthioxanthone (ITX) were purchased from Aladdin Chemistry Co., Ltd. . NIPAAm was recrystallized from hexane and MBAA was purified by recrystallization from acetone before use. All other chemicals were used as received unless otherwise stated. Deionized water was used throughout.A twill cotton fabric  was supplied by Zhejiang Furun Co., Ltd. . Alginic acid sodium (SA), extracted from brown algae  during the hydrogel preparation. The resulting PNIPAAm hydrogel was then immersed in 1% (w/v) CaCl2 aqueous solution at room temperature for 24 h to form the alginate-Ca2+/PNIPAAm IPN hydrogel or alginate-Ca2+/P(NIPAAm-co-EGMA360) IPN hydrogel. Although there are doubts about PNIPAAm and its toxicity and biocompatibility to the human body, the obtained hydrogel in our investigation will be used as wound dressing. Hence, it did not directly go into the human body, and the toxicity of hydrogel can be neglected. To remove the residual monomer, initiator, and cross-linkers in the hydrogel, the obtained hydrogels or fabric grafted hydrogels were placed in distilled water, and the distilled water was exchanged every few hours to extract residual or unreacted chemicals. The reaction mechanism is presented in A series of IPN hydrogels were prepared via free radical polymerization with APS as redox initiator, MBAA as chemical cross-linker, and CaCl360, SA, and other additives were dissolved. UV irradiation was carried out in an ultraviolet processor  at room temperature for 30 min. The processor was equipped with a high-pressure mercury lamp (400 W) with a wavelength range of 320–390 nm. After irradiation, the samples were immersed in 1% (w/v) CaCl2 aqueous solution for 24 h and soaked in distilled water to obtain the final purified cotton fabric grafted hydrogels. The procedure and mechanism of UV photo-grafting method is schematically shown in The UV photo-grafting method was used to prepare cotton fabric grafted hydrogels. In the first step, the cotton fabrics were saturated with acetone solution of 0.1 wt % ITX and exposed to UV light for 8 min to form surface photo-initiators. Then, the pre-treated fabrics were placed on the bottom of the mold, and a proper amount of reaction solution was transferred into the mold, in which the given concentrations of NIPAAm, EGMA−1 and with a resolution of 4 cm−1.ATR-FTIR spectroscopy was used to analyze the chemical structure of these samples. They were dried in a vacuum freeze drier  and were then measured with a Fourier Infrared Spectrometer  in the range of 4000–400 cmThermogravimetric analysis of the hydrogel samples was performed with a thermal gravimetric analyzer (TGA) . 10 mg of dried hydrogel samples was used for each measurement at a 20 °C/min heating rate in the range of 30 to 800 °C and with a 50 mL/min nitrogen flow.Tg) of the hydrogel samples. The hydrogel samples were immersed in deionized water at room temperature and allowed to swell for 48 h to reach equilibrium. The swollen samples were then performed from 25 to 40 °C at a 1 °C/min heating rate to measure the LCST, and the dried hydrogel samples were used for the Tg measurement at 1 °C/min heating rate in the range of −50 °C to 150 °C. The samples for DSC test were around 10 mg, measured in a nitrogen atmosphere.A differential scanning calorimeter  was used to determine the lower critical solution temperature (LCST) and the glass transition temperature (The surface morphology of hydrogel samples was probed by a scanning electron microscope (SEM) . The dried samples were mounted on an aluminum stud using conductive adhesive and sputtered with gold using an auto fine coater .SR) was calculated by Equation (1).tW is the mass of the swollen hydrogel at any given time and dW the mass of the dry hydrogel.Hydrogel samples were freeze dried at −40 °C for 24 h. The dried samples were weighed before being soaked in distilled water and incubated at 25 °C. The swollen samples were moved out from the water at predetermined time intervals, blot dried, and weighed. This procedure was repeated until the weight of swollen samples reached a constant value. The average values among three measurements were taken for each sample, and the swelling ratio (WL%) was defined by Equation (2).eW is the mass of the swollen hydrogel at equilibrium and the other symbols are the same as defined above.The deswelling kinetics of a hydrogel sample was measured gravimetrically by transferring blot-dried hydrogels that were swollen and reached equilibrium at 25 °C to a water bath of 50 °C. The weight change of the hydrogel was recorded in the course of deswelling at different time intervals. 50 °C is well above the LCST of the hydrogels in this study, and dramatic loss of water could be attained within a short timeframe. All deswelling kinetics studies were carried out in triplicate, and water loss percentage , according to the AATCC Test Method 202. Before being measured, the samples were stored in an atmosphere with a temperature of 20 ± 2 °C and a relative humidity of 65 ± 3% for 24 h. During the measurements, the system monitored the resultant force-displacement curve. The curve area indicates the energy needed to wrinkle the fabrics, which means the fabrics with a smaller curve area has better hand feeling.2+/PNIPAAm hydrogel and alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogel were shown in −1 (O–H stretching vibrations), 2900–2800 cm−1 (C–H stretching vibrations), the amide I band , and the amide II band  were identified [−1 (guide line 1) was assigned to the C=O stretching vibrations of PEGMA360, the absorptions at 1383 cm−1 (guide line 2) and 1373 cm−1 (guide line 3) were characteristic peaks of PNIPAAm associated with the isopropyl groups, and the absorbance peaks at 1173 cm−1 (guide line 4) was identified to the C–N stretching vibrations [−1 to 1083 cm−1 (guide line 5) may have been due to the interactions between the ethoxy groups of PEGMA360 and the hydroxyls groups of SA.The ATR-FTIR spectra of alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogels, which exhibited a multistep mass loss behavior. The weight of the alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogels underwent a mass loss approximating to 10% below 100 °C, possibly due to the evaporation of free water. At temperature 250–450 °C, the polymer started to decompose by going through a complex process including degradation of side chains and further depolymerization of the main chain structure. The half-weight loss temperatures for the alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogel (NIPAA:EGMA360 = 20:0) and alginate-Ca2+/PNIPAAm hydrogel (NIPAA:EGMA360 = 18.5:1.5) were 425 °C and 414 °C, respectively. Thus, with an increasing monomer ratio pf EGMA360, alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogels more easily decomposed above 250 °C. However, below 250 °C, the hydrogel with a molar ratio of NIPAAm:EGMA360 = 18.5:1.5 still exhibited an excellent thermal stability and had no effect on their application in daily life.2+/P(NIPAAm-co-EGMA360) hydrogels determined from DSC thermogram is shown in 2+/P(NIPAAm-co-EGMA360) hydrogels with monomers ratios of NIPAAm to EGMA360 of 20:0, 19.5:1.5 and 18.5:2.5 are 34.76, 36.34 and 40.07 °C, respectively. Thus, the LCSTs of hydrogels shit towards higher value with increase of the EGMA360 monomer ratio, which was attributed to the enhanced hydrophilicity. The thermal sensitivity of hydrogels was attributed to their rapid alteration from hydrophilicity to hydrophobicity. Blow the LCST, the hydrophilic groups were bonded to water molecules through hydrogen bonds, and the hydrogels exhibited swollen state in the macroscopic. When the external temperature was increased, the hydrophobic interactions became dominant, leading to shrinkage of the hydrogel [360 into PNIPAAm would increase the hydrophilicity of the hydrogel network due to the hydrophilicity of EGMA360. Therefore, the hydrogen bonds between the hydroxyl group in the side chain of EGMA360 and the water molecules would be significantly enhanced below the LCST, and higher temperature is required to break these hydrogen bonds.The LCST of alginate-Cahydrogel ,30,31. T2+/P(NIPAAm-co-EGMA360) hydrogels, the effect of temperature on the water retention capacity of the hydrogels was evaluated by measuring their swelling ratios under external stimuli. The temperature dependent equilibrium swelling ratios in the range from 25 to 60 °C were studied, and the experiment was repeated thrice for each sample and the average values were used in data analysis. As shown in 360 ratio, which fit to the result obtained from the DSC thermograms analysis  hydrogels was also measured with DSC . The measured Tgs of alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogels with monomer ratios of 19.5:0.5 and 18.5:1.5 are 66.44 °C and 14.49 °C, respectively. Thus, Tg is dramatically reduced by increasing the amount of EGMA360 in the hydrogels. The EGMA360 applied in this study contains 4 or 5 ethoxy groups in the side chain and is regarded as a flexible segment. For this reason, the flexibility of the hydrogel is enhanced after introducing EGMA360, which leads to the decrease of Tg.The influence of the EGMAwith DSC . The Tg The thermal response of hydrogel to external temperatures was captured by a digital camera under a microscope and presented in 360 molar ratios were measured by immersing the hydrogels in water at 25 °C for 48 h  possessed the highest swelling ratio (25.22 g/g), while that of alginate-Ca2+/PNIPAAm hydrogels (NIPAAm:EGMA360 = 20:0) was the lowest (20.25 g/g). The swelling ratios of these hydrogels were mainly related to the hydrophilicity. The additional hydroxyl group at the end of the EGMA360 side chain will enhance the hydrophilicity of the hydrogels. For this reason, more PEGMA360 component in the hydrogel structure facilitated the hydration and expansion of the hydrogels. In addition, the porous structure of the IPNs greatly improved the diffusion of water into the hydrogel network. Combining these two factors, the swelling capability of the hydrogels with EGMA360 is remarkably increased. In our initial experiment, SA and NIPAAm were applied as the monomers, MBAA as chemical cross-linker, APS as initiator, TEMED as catalyst, and CaCl2 as physical cross-linker to prepared alginate-Ca2+/PNIPAAm IPN hydrogels. Afterward, EGMA360 was further introduced into the alginate-Ca2+/PNIPAAm networks to form IPN in the later investigation. In order to minimize the effects of the cross-linker to our investigation of the influence of EGMA360 to the hydration and transition behaviors, MBAA was still used as the cross-linker in the present investigation. In our future study, we will also choose ethylene glycol dimethacrylate (EGDMA) as the cross-linker to prepare alginate-Ca2+/P(NIPAAm-co-EGMA) hydrogels, hence the effect of a different cross-linker to the swelling and transition behaviors of hydrogels can be assessed.The swelling behaviors of hydrogels with different NIPAAm/EGMAfor 48 h a. In gentW is the mass of swollen hydrogel at a given time, eW is the mass of equilibrium swelling hydrogel, t is the diffusion time, k is a constant, and n is the diffusion index. When n ≤ 0.5 or n ≥ 1, the diffusion kinetics obey Fick or non-Fick diffusion, respectively, whereas the anomalous diffusion refers to the case when 0.5 < n <1 [n value by determining the slope of the plot ln(tW/eW) against lnt.In order to understand the swelling kinetics of hydrogels, water diffusion in the hydrogels was analyzed by fitting the swelling data into the power law equation (diffusion/relaxation model) ,33,34, a5 < n <1 ,36. EquatW/eW) as a function of lnt was plotted in n values are obviously influenced by EGMA360 component, which ranged from 0.37, 0.57 to 0.56 with monomer ratios of 20:0, 19.5:0.5, and 18.5:1.5 (NIPAAm:EGMA360). It indicated that introduction of EGMA induced the shift of diffusion from a Fickian model to an anomalous model.ln hydrogels by losing a larger volume of water in same time frame. Being kept in 50 °C water for about 20 min, the alginate-Ca2+/PNIPAAm hydrogels shrank and lost 68% water, while alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogels still held about 50% water. On the other hand, the ultimate water loss percentage of the alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogels slightly decreases with the increase of EGMA360 ratio. It is well known that when the temperature is above the LCST, the thermo-responsive polymer switches from the hydrophilic state to hydrophobic state. Thus, the shrinkage of the hydrogels sets in. For the IPN hydrogels, EGMA360 was introduced into the alginate-Ca2+/PNIPAAm networks to form IPN, the hydrophilcity of the hydroxyl groups at the side chain of PEGMA360 is better than that of acylamino groups in PNIPAAm, resulting in relative difficultly removing of water molecules from the gel matrix. Thus, the shrinking rates were slightly weakened.The deswelling kinetics of the hydrogels are shown in k is the shrinkage constant, and a greater k means a faster shrinkage process [To quantitatively analyze the deswelling behavior, a semi-logarithmic plot as a first ordered rate analysis was applied to the time dependence deswelling kinetics analysis ,38.(5)l process .360 = 20:0) provides the largest shrinkage rate constant (k = 0.01207), followed by the one with NIPAAm:EGMA360 = 19.5:0.5 (k = 0.01195) and 18.5:1.5 (k = 0.01055). The smaller value of k is an indicator of lower release rate, which agrees with the results of deswelling kinetic curves in 2+/P(NIPAAm-co-EGMA360) IPN hydrogel on cotton fabrics by photo-grafting was confirmed by ATR-FTIR measurements (−1), C–H stretching vibrations (3000–2800 cm−1), and C–O stretching vibrations (1057 cm−1). After grafting alginate-Ca2+/P(NIPAAm-co-EGMA360) IPN hydrogel to the cotton fabrics, all the characteristic peaks  observed in alginate-Ca2+/P(NIPAAm-co-EGMA360) IPN hydrogel spectra (2+/P(NIPAAm-co-EGMA360) IPN hydrogel on the surface of cotton fabrics.Immobilization of alginate-Caurements a. The bl spectra  are all 2+/P(NIPAAm-co-EGMA360) hydrogels in swollen condition (swollen hydrogels were freeze-dried before SEM examination) is shown in The morphology of cotton fabric–grafted alginate-Ca360. The hydrogels exhibited numerous interconnected pore structure. Moreover the pore of these honeycomb-like structures were enlarged with an increase of EGMA360 content in the IPN hydrogel composition, i.e., the hydrogels of NIPAAm:EGMA360 = 18.5:1.5 had the largest pore size, while that of NIPAAm:EGMA360 = 20:0 had the smallest. The results may be attributed to the highly expanded network formed during the crosslinking polymerization process due to the electrostatic repulsions among the hydroxyl (–O−) from EGMA360 and the carboxylates (–COO−) from SA [360 to hydrogel influenced the crosslinking density as well as the hydrophilic/hydrophobic balance of alginate-Ca2+/PNIPAAm hydrogels. Hence, the microstructure of hydrogels experienced a significant change.The data clearly illustrated the dependence of hydrogel morphology on the ratio of NIPAAm to EGMA from SA . The int2+/P(NIPAAm-co-EGMA360) hydrogels presented a typical sudden shrinkage of swelling ratio at 35.58, 36.94 and 42.43 °C (2+/P(NIPAAm-co-EGMA360) hydrogels. This phenomenon indicates that the incorporation of cotton fabrics has little influence on the thermal transition of hydrogels.The thermo-responsive property of the fabric grafted hydrogels was analyzed by measuring their swelling ratios in different temperatures . There w42.43 °C b, which 2+/P(NIPAAm-co-EGMA360) hydrogels with NIPAAm:EGMA360 = 19.5:0.5 (28.07) and 18.5:1.5 (24.38) exhibit no prominent difference. Hence, it can be concluded that the grafted alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogels do not affect the hand feeling of the cotton fabrics. On the contrary, the cotton fabrics with alginate-Ca2+/PNIPAAm hydrogels shows a much larger value (85.57). The huge difference among these curve areas would be attributed to the decrease of Tg value . As the flexibility of hydrogel is enhanced when the Tg is decreased, the hand feeling of the fabrics with hydrogel is also improved.Extraction curve area indicates the energy needed to wrinkle the fabrics, which can be obtained from Phabrometer Fabric System. As the value is inversely proportional to hand values, it can be used to evaluate the hand feeling of the fabrics. Generally, the smaller the curve area, the better the hand feeling of the fabric. 360) to the hydration and transition behaviors of a thermo-responsive interpenetrating network (IPN) hydrogels series consisting of sodium alginate, N-isopropylacrylamide (NIPAAm) and ethylene glycol methacrylate (EGMA360) were investigated. Polymerization of the resulting IPN hydrogels was confirmed by ATR-FTIR and TGA analyses. DSC and temperature stimulus-responsive equilibrium swelling ratio results indicated that hydrogels with increased EGMA360 composition ratio exhibit higher LCST. Additionally, Tg was dramatically shifted towards lower temperatures due the flexible ethoxy groups in EGMA360. Besides, the swelling/deswelling behaviors indicated that a faster water uptake rate and a slower water loss rate appeared with increase of EGMA360 amount. The obtained IPN hydrogels were grafted to the cotton fabric by UV photo-grafting method. SEM was utilized to investigate the morphology and the results demonstrated the porous structure of hydrogel layers. Moreover, the pores of the hydrogels were enlarged with larger amount of EGMA360. Further, the equilibrium swelling ratio of the fabric grafted hydrogels in water decreased upon heating, which confirmed the thermo-responsive property of fabrics with hydrogels. Unlike the fabrics grafted with alginate-Ca2+/PNIPAAm hydrogel, the cotton fabric grafted alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogels present similar hand feeling to the original cotton fabrics. Therefore, alginate-Ca2+/P(NIPAAm-co-EGMA360) hydrogel is a promising candidate for applications in the field of biomedical textiles, such as the wound dressings.In the present work, the influence of ethylene glycol methacrylate (EGMA


To improve the degradation ability of anaerobic granular sludge (AGS) toward the iodinated contrast media (ICM) iohexol, biogenic nanoscale palladium (Pd) was formed in AGS via microbial reduction. The Pd hosted in AGS (Pd-AGS) was used for iohexol degradation. The effects of the electron donor, reaction medium, iodide ion fouling, and polymer embedding of the Pd-AGS on the reactivity were investigated. Our results showed the Pd-AGS increased the degradation rate of iohexol, with a conversion rate constant increased by 86.3-fold compared to the AGS control. Various organic compounds were investigated as electron donors to initiate the catalytic activity of Pd-AGS and the promotion achieved with the tested electron donors was in the following order: formate > lactate > ethanol > glucose > acetate. The Pd-AGS had high reactivity in deionized water at mild pH, and almost no reactivity under acidic (pH = 1.2) and alkaline (pH > 11) conditions. The presence of iodide ions in the medium inhibited the catalytic activity of Pd-AGS toward iohexol because of catalyst fouling. Embedding the Pd-AGS in alginate, chitosan, or polyvinyl alcohol (PVA) could prevent Pd loss but it also retarded the iohexol degradation rate. The Pd-AGS, as a combination of Pd catalyst and AGS, provides a novel strategy for iohexol degradation in polluted water and wastewater. Iodinated contrast media (ICM) are a widely used class of pharmaceuticals for imaging in radiological and medical diagnoses . ICM comMany methods have been investigated to enhance ICM degradation, such as ultraviolet-based advanced oxidation , sulfatePalladium nanoparticles are often fabricated using chemical methods with lots of chemicals under harsh conditions. Preparation of PdNPs through microbial reduction is regarded as a promising and environmentally friendly method as it uses less toxic chemicals and no stabilizers or carriers . Unlike In this study, Bio-Pd was prepared using AGS as the Pd (II) reducer and immobilization carrier. The reaction activities of the Pd-AGS toward iohexol were evaluated with different electron donors, reaction mediums, and immobilization methods. This study provides detailed information on the application of Pd-AGS to degradation of ICM.Supplementary Figure S1) was purchased from Sigma-Aldrich (purity >95%). The AGS for Bio-Pd production was obtained from a full-scale anaerobic reactor treating wastewater from beer production in Beijing, China. The AGS had a mean diameter of approximately 5 mm and good settling ability with a Sludge Volume Index of around 20. Unless otherwise stated, all the reagents used in this study were of analytical grade.Iohexol . After being tightly sealed with inert Viton stoppers, the bottles were incubated in a shaker (180 rpm) at 35 ± 1°C for 8 h to reduce Pd (II). The Pd-AGS in the reaction medium was subsequently collected by centrifugation at 3000 × g for 10 min, and then gently washed with distilled water three times in preparation for use in the iohexol reduction experiments. The PdNPs can be formed in the cell walls, periplasmic space, or cytoplasm of microbes of AGS  for 10 min to remove oxygen, and finally tightly sealed with stoppers and incubated on a shaker (180 rpm) at 35 ± 1°C. Samples were withdrawn after specific intervals and the concentration of iohexol remaining in each sample was measured.Degradation of iohexol by the Pd-AGS was assessed in the presence of various electron donors . First, Pd-AGS or AGS control pellets  were added to serum bottles . Then, the reaction medium and electron donor (each at 500 mg/L), and iohexol stock solution  were added. The bottles were flushed with N2SO4, or an alkaline medium containing Na2CO3 (pH = 11.2) or NaOH (pH = 11.7).To investigate the effects of the reaction medium on iohexol degradation by Pd-AGS, similar degradation experiments were conducted in deionized (DI) water (pH = 6.9), phosphate buffered saline , an acidic medium (pH = 1.2) spiked with HIn addition, the effect of iodide ions on iohexol degradation by the Pd-AGS was investigated with various iodide ion concentrations. We used iodide ion concentrations of 0.073, 3.65, 7.3, and 36.5 mM, which corresponded to 1, 50, 100, and 500 times the theoretical iodide ion concentration released from 20 mg/L iohexol, respectively.2 with a syringe to yield PVA or alginate gel beads, or dropped into glutaraldehyde (0.5%) solution containing 2% CaCl2 to yield chitosan gel beads. Crosslinking at 25 ± 1°C for 24 h generated gel beads with a diameter of 5–7 mm. These gel beads were then washed and used for iohexol degradation.To prevent Bio-Pd loss from Pd-AGS, we attempted to embed the Pd-AGS in sodium alginate, chitosan, or polyvinyl alcohol (PVA) using the following procedure. First, the Pd-AGS was collected and gently washed with a PBS solution three times. Chitosan was dissolved in an acetic acid solution (2.5% v/v) to make a 5% (w/v) chitosan solution. Sodium alginate was dissolved in distilled water to make a 3% (w/v) alginate solution. A solution of PVA (10% w/v) was made by dissolving PVA in distilled water. The AGS or Pd-AGS  was then added to the above gel solutions and mixed completely. The gel–biomass mixture was then dropped into a saturated boric acid solution containing 2% CaClThe concentration of iohexol was analyzed using an ultra-performance liquid chromatography  with a 2996 photodiode array detector at a detection wavelength of 237 nm. The mobile phase was a mixture of methanol and water (7:93) (v/v) at a flow rate of 0.3 mL/min. Iodide ion analysis was performed by ion chromatography  using 35 mM NaOH for elution. Total organic carbon (TOC) was measured using a TOC-V analyzer .Figure 1). Only 11% of the iohexol was removed by the AGS control after 12 h in the presence of formate. With Pd-AGS, 51–100% of the iohexol was removed in the presence of the tested electron donors. These data indicate that combination of Bio-Pd with AGS greatly increased iohexol degradation. Except for acetate, all of the tested electron donors promoted iohexol degradation. The resulting data were well fitted to a pseudo-first order kinetics model based on the following equations:The Pd-based HDC process requires hydrogen and electron donors to initiate the catalyst. The effects of various electron donors  on the reactivity of Pd-AGS toward iohexol were investigated (andt is the concentration of iohexol (mg/L) at time t, [IOX]0 is the initial concentration of IOX (mg/L), and obsK is the observed first-order rate constant (min−1). Conversion rate constants were obtained with different electron donors (Table 1).where [IOX]obsK (0.0135–0.0349 min−1) than the AGS control (0.0004 min−1), with obsK increased by 32- to 86-fold. Compared to the experiment conducted with no electron donor, the obsK for the Pd-AGS increased by factors of 13.4, 7.6, 5.2, and 4.1 in the presence of formate, lactate, ethanol, and glucose, respectively. Iohexol was degraded to a certain extent even without the presence of an electron donor, possibly because some microbial lysis or digestion products released from the Pd-AGS could serve as electron donors to initiate the Pd activity. The promotion achieved with the tested electron donors was in the following order: formate > lactate > ethanol > glucose > acetate. We previously investigated azo dye reduction using Pd-AGS in the presence of various electron donors, and obtained similar results to the present study . The iohexol conversion rate constant increased from 0.0036 to 0.0046 min−1 when the formate concentration was increased from 500 to 2000 mg/L. Therefore, sufficient formate is needed to obtain a high iohexol conversion rate.Formate was most effective electron donor for iohexol conversion by Pd-AGS in this study. The effects of the formate concentration on the reactivity of Pd-AGS were further investigated . During the first 3 h, the iodide ion release rate (85%) was slightly lower than the iohexol removal rate (93%), possibly because the initial adsorption of iohexol on the surface of Pd-AGS may contribute partially to its removal. After 8 h, both the iohexol removal and iodide ion release rate reached 95% . The C-I bond is weaker than a C-Br, C-Cl, or C-F bond, which may be a reason for the high release rate of iodide ions from iohexol degraded by Pd-AGS , suggesting adsorption and biodegradation by the AGS were not the primary reasons for iohexol removal. The removal of TOC was also measured during the iohexol degradation by Pd-AGS (data not shown), and less than 15% was removed at the end of reaction. Based on these data, it could be deduced that deiodination of iohexol through catalytic reduction by Pd catalyst was the main mechanism for iohexol removal by the Pd-AGS.Iodide ion release during iohexol degradation by Pd-AGS was investigated further. Iohexol was degraded rapidly at the beginning of the reaction, and this was accompanied by rapid iodide ion release . The initial iohexol degradation rates were 2.771, 6.231, and 9.587 mg/(L⋅h) with iohexol concentrations of 20, 50, and 100 mg/L, respectively. Increasing the initial concentration of iohexol led to an increased initial degradation rate, indicating a positive correlation between the adsorption of iohexol on the Pd-AGS and the Pd-AGS catalytic activity. This dependence was clarified by fitting the experimental data to the Langmuir–Hinshelwood model  of 0.99. This result indicated that the catalytic degradation of iohexol by the Pd-AGS followed the Langmuir–Hinshelwood model and the conversion of adsorbed iohexol on the catalyst surface was the rate controlling step.where Figure 6). The highest iohexol degradation rate was obtained in DI water (pH = 6.9), with a kinetic rate constant of 0.0034 ± 0.0002 min−1. In PBS (pH = 7.5), the degradation rate of iohexol was slightly retarded compared with that in DI water and the rate constant decreased to 0.0019 ± 0.0001 min−1. This retardation could be caused by Pd poisoning by phosphate ions (2CO3 (pH = 11.2), the degradation of iohexol was significantly inhibited (p < 0.05) and the rate constant decreased to 0.0009 ± 0.00005 and 0.0007 ± 0.00003 min−1, respectively. This could occur because the presence of OH− could inhibit the reactivity of the Pd catalyst by blocking Pdn+ active sites . The results showed that the presence of iodide ions retarded iohexol degradation significantly (p < 0.05), and the kinetic rate constant decreased by 45–73% compared with that in the DI water . Iodide ions have a strong affinity to the surface of the Pd catalyst and can block its active sites, which may contribute to inhibition of the HDC reaction . In most cases, the iodide concentration in water or wastewater is much lower than the concentration tested in this study and the iodide ion fouled Pd catalyst can be restored through DI water washing.Released halide ions may influence the HDC process with a Pd catalyst. Degradation of iohexol by Pd-AGS was investigated in the presence of iodide ions at various concentrations  . Immobilization using chitosan, alginate, and PVA decreased the catalytic activity, and kinetic rate constant decreased to 18.9, 9.3, and 8.4%, respectively, of the original Pd-AGS. Immobilization using a polymer adds a polymer layer around the Pd-AGS and can retard diffusion and transfer of substances between the AGS and aqueous phase. In addition, the active sites of the Pd catalyst may be covered by the polymer or fouled by other organic or inorganic species during the immobilization process. All these factors may contribute to loss of catalytic activity after polymer immobilization. Therefore, polymer immobilization is not suggested as a stabilization strategy for Pd-AGS during application.As some microbes in AGS may suffer toxicity from Pd (II) during reduction process and therefore detach from AGS, we investigated embedding the Pd-AGS in polymer alginate, chitosan, or PVA to prevent the biomass and catalyst loss. The effects of immobilization on the Pd-AGS reactivity were investigated  was achieved using the Pd-AGS within 2 h in the presence of formate as the electron donor. As a combination of a Pd catalyst with a live microbial culture, a variety of organic compounds could serve as the electron donor to initiate the catalytic activity of Pd-AGS. The efficiency of the tested electron donors was in the following order: formate > lactate > ethanol > glucose > acetate. The removal of iohexol followed the Langmuir–Hinshelwood model and the conversion of adsorbed iohexol on the surface of Pd-AGS was the rate controlling step. The Pd-AGS had high reactivity in DI water at mild pH, and almost no reactivity in acidic (pH = 1.2) and alkaline (pH > 11) media. The presence of iodide ion in the medium also reduced the Pd-AGS catalytic activity. Polymer immobilization is not suggested for the Pd-AGS as it may greatly decrease the reactivity. The Pd-AGS provides a novel strategy for iohexol removal from slightly polluted water.Biogenic PdNPs were XQ designed the experiments and wrote the manuscript. XZ and YS performed the experiments and data analysis. JZ provided analytical support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To objectively evaluate posterior capsular opacification (PCO) with RTVue-100 spectral domain-OCT and assess the agreement with the Pentacam system.  Sixty-seven eyes diagnosed with PCO were included. RTVue-100 SD-OCT was used to scan the IOL outline and PCO at horizontal and vertical meridians. PCO was also imaged with a Pentacam and slit-lamp photography system. With RTVue-100 SD-OCT, the PCO area, thickness, density, and objective scores were recorded and used to evaluate the severity of PCO at 3 mm and 5 mm diameter ranges of the IOL optic region. We assessed the correlation of visual acuity, PCO characteristics, and PCO scores. PCO scores acquired from RTVue-100 SD-OCT images were also compared with those from the Pentacam. Differences between pear-type and fibrosis-type PCOs were also compared using RTVue-100 SD-OCT cross-sectional images.  The cross-sectional images of PCO acquired with RTVue-100 SD-OCT corresponded well to Pentacam and slit-lamp retroillumination images. IOL-posterior capsular space, area, thickness, and density of the proliferated and accumulated LECs could be clearly visualized and quantified with RTVue-100 SD-OCT. PCO scores were correlated with decreased visual acuity, which was in line with the outcomes using the Pentacam. Differences between the pear-type and fibrosis-type PCO were statistically significant; pear-type PCOs showed a wider and thicker opacification region with lower density compared with fibrosis-type PCOs.  RTVue-100 SD-OCT could be a powerful tool in PCO objective evaluation and classification. OCT could be used to visualize the morphology and outline of PCO. Thus, it could discriminate and quantify differences between different types of PCO. PCO scores seem to be a useful factor that could reliably reflect PCO severity. Posterior capsular opacification (PCO) is one of the most common complications after cataract surgery, which can be treated with neodymium-yttrium-aluminium-garnet (Nd:YAG) laser capsulotomy. However, the increased cost of treatment, risk of retinal detachment , and macOCT is a noninvasive technique based on low-coherence interferometry that could generate high-resolution cross-sectional images from backscattered light, thus benefiting ophthalmologists in evaluating different retinal diseases and assessing changes in the anterior segment of the eye , 10. It However, PCO quantification was necessary and meaningful. RTVue-100 SD-OCT could provide detailed information on the cloudy posterior capsule with cross-sectional images, which allows precise evaluation of the opacification area, thickness, and density. Even the area of IOL-posterior capsular space could be quantified.We therefore aimed to assess the application of RTVue-100 OCT in a PCO severity quantification study and compare the images among RTVue-100 OCT, Pentacam, and slit-lamp methods in PCO evaluation and classification.This prospective observational study recruited 67 patients (67 eyes) implanted with a posterior capsular IOL who had been diagnosed with PCO and planned to receive Nd:YAG capsulotomy surgery. The study was approved by the Research Review Board and strictly followed the tenets of the Declaration of Helsinki (1989) of the World Medical Association. Informed consent was collected before Nd:YAG capsulotomy surgery. A total of 10 eyes of 10 patients received uneventful phacoemulsification and IOL implant surgery without PCO and were enrolled as controls.Routine examinations were done by an expert ophthalmologist. Exclusion criteria were as follows: cornea opacity, uveitis, glaucoma, eventful cataract surgery, eye with poor fixation ability, a history of eye trauma and complicated surgery, and a history of severe systemic disease. Visual acuity (VA) was recorded using a Snellen E chart before mydriasis and was converted to the logarithm of the minimum angle of resolution (logMAR) value for statistical analysis. Retroillumination photography, Pentacam images, and RTVue-100 OCT images were acquired after pupil dilation.Retroillumination photographs of the anterior segment, especially the posterior capsular region, were obtained with the slit-lamp photograph system  after pupil dilation.A Pentacam system  was used in combination with the Scheimpflug video-photography system and a monochromatic slit-light source that provided a 3-dimensional scan of the anterior segment of the eye. In brief, the Scheimpflug slit images of the anterior segment were taken with a rotating camera from 0 to 360 degree meridians after pupil dilation. Twenty-five images were taken in 2 seconds, and eye movement was automatically corrected during imaging processing. Patients were told to not blink their eyes during measurement. After measurement, the high-quality images at the horizontal and vertical meridians were selected and transferred to a computer for further analysis. The area and density of PCO at a diameter of 5 mm within the IOL optic region were evaluated using Image Pro Plus software 6.0. The opacification density values were expressed as computer compatible tape, and the scattered light intensity ranged from 0 to 255 . PCO denPCO score definition: PCO score = PCO area × (PCO density−scatter light intensity)μm. A corneal anterior module long adaptor lens was mounted on the probe and focused on the anterior segment part, including the IOL and PCO after pupil dilation. Patients were told to fixate straight ahead on the red light using the fellow eye [RTVue-100 OCT , based on the spectral domain technique with a corneal anterior module long adaptor lens (1.96 mm scan depth and 6 mm scan width), was adopted in our study. A super luminescence diode emitted light with a 50 nm bandwidth was centred at 830 nm and had a scan speed of 26,000 axial scans per second with an axial resolution of 5 llow eye . Using aU test was used to compare differences between pear-like PCO and fibrosis-type PCO. p values less than 0.05 were considered significant.Statistical analyses were performed with IBM SPSS Statistics 23.0. The Kolmogorov–Smirnov test was used to assess the distribution of all data. A nonparametric Spearman test was used to evaluate the relationship among visual acuity, PCO characteristics , and IOL-posterior capsular spaces measured with RTVue-100 OCT, as well as the relationship between PCO characteristics measured with RTVue-100 OCT and the Pentacam. A Mann–Whitney The study analysed PCO characteristics of 67 pseudophakic eyes measured with RTVue-100 OCT. The average age of the patients was 66.91 ± 11.10 (ranging from 37 to 91) years old. The average PCO formation time was 35.79 ± 24.15 months. The average visual acuity was 0.40 ± 0.11 (ranging from 0.15 to 0.52).PCO could be clearly visualized and classified by morphology with images measured by RTVue-100 OCT (2. The density of the IOL-posterior capsular space was 32.05 ± 14.12 (6.26 to 99.78), which was significantly correlated with decreased visual acuity , as described in PCO characteristics measured with RTVue-100 OCT are shown in r=0.43, p ≤ 0.001) and 5 mm  diameters of the IOL optic region. For pear-like cases, the correlation efficient of PCO score was 0.36 (p=0.01) and the fibrosis PCO was 0.56 (p=0.04) at the 3 mm diameter of the IOL optic region  diameters of the IOL optic regions  were correlated with those from RTVue-100 OCT both at the 3 mm and 5 mm . The PCO area, thickness, and density at 3 mm  of the IOL optic region were significantly different, as well as the PCO at the 5 mm IOL optic region. PCO scores were 23.57 ± 7.78 for the pear-like type and 16.94 + 11.99 for the fibrosis type, and these differences were not statistically significant at the 3 mm IOL optic region . Details of comparisons between fibrosis PCO and pear-like PCO are described in In comparisons between fibrosis PCO and pear-like PCO, the central distance and full area of the IOL-posterior capsular spaces were significantly different , contrast sensitivity, and stray light will be the focus of a future study. Third, RTVue-100 OCT and Pentacam images were cross-sectional images of the IOL and PCO, although they were well in line with the retroillumination images and decreased visual acuity. Therefore, comparison studies between RTVue-100 OCT and other PCO evaluation methods will also be useful.In summary, by using RTVue-100 OCT, we could clearly observe the IOL, posterior capsule, and the accumulated extracellular substance and LECs. The objective quantification of the PCO area, thickness, and density demonstrated a correlation between decreased visual acuity and IOL-posterior capsular space. The new objectively defined PCO score was in line with the PCO score evaluated with the Pentacam system and was correlated with the width of the IOL-posterior capsular space and the decreased visual acuity. Differences between the pear-type and fibrosis-type PCOs could also be clearly distinguished. These results suggested that RTVue-100 OCT could be used as a powerful method to evaluate the IOL-posterior capsular space and the classification and quantification of PCO.


The results showed that hypoxia and drug treatment could both induce a significant reduction in the TER of the intestinal epithelial cell monolayer and a significant reduction in the expression of the ZO-1 and occludin protein. Hypoxia and LatA could cause a significant reduction in the ratio of F-actin/G-actin content, whereas jasplakinolide caused a significant increase in the ratio of F-actin/G-actin content. After hypoxia, cofilin phosphorylation was decreased. We concluded that the barrier function of the intestinal epithelial cell monolayer was significantly damaged after severe burn injury. The molecular mechanism might be that hypoxia-induced F-actin depolymerization and an imbalance between F-actin and G-actin through cofilin activation resulted in reduced expression and a change in the distribution of cellular TJ proteins.Mechanical barriers play a key role in maintaining the normal function of the intestinal mucosa. The barrier function of intestinal epithelial cells is significantly damaged after severe hypoxia. However, the molecular mechanisms underlying this hypoxia-induced damage are still not completely clear. Through the establishment of an  Their bThe Caco-2 cell line was purchased from ATCC. Dulbecco’s modified Eagle medium (DMEM), nonessential amino acid (NEAA), and fetal bovine serum (FBS) were purchased from GIBCO , and cell culture incubator and multiplate reader were purchased from Thermo Fisher . Trypsin was purchased from BBI Life Sciences Corporation . Twenty-four-well plate Transwell chambers (8 mm per well) were purchased from Corning . The Alexa Fluor 488-deoxyribonuclease and Alexa Fluor 594-phalloidin were purchased from Invitrogen . TCS SP5 laser confocal microscope was purchased from Leica (Germany). Anti-β-actin, anti-cofilin, anti-phosphorylated cofilin, anti-ZO-1, anti-occludin, and FITC-dextran fluorescence probe were purchased from Sigma-Aldrich . Polyvinylidene fluoride (PVDF) membrane and the Millicell ERS voltohmmeter were purchased from Millipore . Jasplakinolide and latrunculin A (LatA) were purchased from Calbiochem (United States). UD-201 tissue and cell ultrasonicator were purchased from Tomy (Japan). RC DC protein assay kit, protein assay kit, protein electrophoresis reagents, electrophoresis cell, electroporation devices, and ChemiDoc XRS gel imaging system were purchased from Bio-Rad Laboratories .2 and saturated humidity. The medium was replaced every other day until 80% confluence, followed by partial digestion with Ca2+-free and Mg2+-free Hank’s balanced saline solution containing 0.25% trypsin and 0.53 mmol/L EDTA. The cells were subcultured in a ratio of 1:3.The Caco-2 cells were cultured in DMEM (pH 7.4), which contains 100 U/ml penicillin, 100 mg/ml streptomycin, 10% FBS, 2 mmol/L glutamine, and 1 mmol/L NEAA at 37°C in an incubator containing 5% CO4 cell/cm2, onto coverslips in 24-well plates at a density of 2.5 × 105 cell/cm2, and into six-well plates at a density of 5 × 105 cell/cm2. The conditions of cell growth in six-well plates, on coverslips in 24-well plates, and in 96-well plates were observed every day under an inverted microscope.The cells were inoculated into 96-well plates and onto the polycarbonate membrane cell culture chambers of 24-well Transwell plates at a density of 5 × 102, 5% CO2, and a 94% N2 mixed gas environment.Caco-2 cells were seeded overnight in plates, followed by treatment with hypoxia. They were randomly divided into six groups using a random number table. Hypoxia was induced according to previously published methods . Cells wCaco-2 cells were seeded overnight in plates, followed by treatment with drugs. They were randomly divided into six groups using a random number table. Jasplakinolide was prepared at a final concentration of 0.1 μmol/L and was added to the cells for treatments for 0 (no treatment), 0.25, 0.5, 1, 2, 6, and 24 h. Using the same method, Lat A was prepared at a final concentration of 0.2 μmol/L and was added to the cells for treatment for 0 (immediately), 0.25, 0.5, 1, 2, 6, and 24 h.The Caco-2 cells were seeded in 24-well Transwell plates to grow as monolayers. After hypoxia and drug treatments, the Millicell ERS voltohmmeter  was used to measure the TER. To clarify the difference of TER between treatment groups and nontreatment group (0 h) more clearly, the normalization method was used to express TER ratio of treatment group to nontreatment group (0 h).FITC-dextran fluorescence probe tracing was used to detect the permeability of the Caco-2 cell monolayers pretreated with drugs for 24 h. The specific manipulation was as follows: the culture medium in the Transwell was discarded, and the upper and lower chambers of the Transwell were washed using D-Hank’s solution. Next, 500 μl of D-Hank’s solution was added into the lower chamber, and 100 μl of the FITC-dextran solution was added into the upper chamber. Cells were routinely cultured for 2 h in an incubator. The fluorescence intensity of the solution in the lower chamber was detected using a multifunctional plate reader with excitation and emission wavelengths of 490 and 520 nm, respectively. The normalization method was used to express the fluorescence intensity ratio of treatment group to nontreatment group (N).2O for 10 s, and placed onto a slide. Next, 2.5 μl of Slowfade solution was dropped onto the slide and covered with a coverslip. The slide was observed under a TCS SP5 laser scanning confocal microscope and photographed.Caco-2 cells were collected at different time points after hypoxia treatment. The culture medium was discarded, and PBS was added into the 24-well Transwell and washed for 5 min. The PBS was then discarded, and an appropriate amount of 4% paraformaldehyde was added for fixation at the room temperature for 30 min. The polycarbonate membrane of the Transwell was washed with PBS for 5 min and rinsed in PBS containing 50 mmol/L NH4Cl at room temperature for 15 min. The membrane was then washed three times for 5 min with PBS. Next, 2.5% BSA (prepared in PBS) was added for blocking at room temperature for 30 min. The PBS was discarded, and the primary anti-ZO-1 antibody at a dilution of 1:200 or the primary anti-occludin antibody at a dilution of 1:100 was added in 50 μl/well and incubated overnight in a 4°C refrigerator. The membrane was then washed again with PBS . DAPI stain at a dilution of 1:2,000 mixed with a Texas red-labeled secondary antibody at a dilution of 1:50 or an Alexa Fluor 488-labeled secondary antibody at a dilution of 1:100 was added in 50 μl/well and incubated at room temperature for 1 h in the dark. The membrane was then washed with PBS . The polycarbonate Transwell membrane was cut along the edge using a razor blade, rinsed in ddHCaco-2 cells inoculated onto 96-well plates were collected at different time points after hypoxia. The culture medium in the original wells was discarded, and the Caco-2 cells in the 96-well plates were washed with PBS . The PBS was discarded, and an appropriate amount of 4% paraformaldehyde was added for fixation at the room temperature for 15 min. The cells were then washed with PBS . Next, 0.1% Triton X-100 was added, and the cells were incubated for 15 min at room temperature, after which 25 g/L BSA was added for blocking, and the cells were incubated at room temperature for 30 min. The solution was discarded, and the cells were washed with PBS . Alexa Fluor 594-phalloidin  and Alexa Fluor 488-DNase I  were added in 50 μl/well and incubated for 15 min to perform specific fluorescence labeling of F-actin and G-actin, respectively. Cells were washed with PBS again . The PBS was discarded, and the red fluorescence intensity (reflecting the relative F-actin levels) and green fluorescence intensity (reflecting the relative G-actin levels) were detected with excitation and emission wavelengths of 578 and 496 nm, and 600 and 519 nm, respectively. The ratio between the relative levels of F-actin and G-actin was the ratio between the intensity of red and green fluorescence.Caco-2 cells in plates were collected at different time points after hypoxia treatment. The culture medium in the original wells was discarded, and the Caco-2 cells on coverslips were washed with PBS for 5 min. The PBS was discarded, and the Caco-2 cells were fixed in 4% paraformaldehyde at the room temperature for 15 min. The Caco-2 cells on coverslips were washed with PBS , and 0.1% Triton X-100 was added, followed by incubation at room temperature for 15 min. Next, 25 g/L BSA was added for blocking, and the cells were incubated at room temperature for 30 min. The BSA was discarded, and the coverslips were washed with PBS . Alexa Fluor 594-phalloidin  was added in 50 μl/well and incubated at room temperature for 15 min. The coverslips were then washed with PBS . An appropriate amount of anti-fluorescence fade reagent was dropped onto the coverslips, and they were placed onto slides with the Caco-2 cells facing downward. Slides were observed under a laser scanning confocal microscope.These experiments were performed according to previously published methods using β-actin as an internal control . Brieflyt test. The comparisons among groups were performed using the one-way analysis of variance (ANOVA). p < 0.05 indicated that the difference was significant and had statistical significance.SPSS17.0  and Excel 2013  software were used for statistical analysis. Data are expressed as the mean ± SD. The comparison of mean values between two samples was performed using the It is known that TER is a marker of epithelial barrier function. As shown in Altered tight junction structure often contributes to the impaired epithelial barrier function. We further explored the effect of hypoxia on the distribution of the TJ proteins ZO-1 and occludin using immunofluorescence combined with confocal microscopy. The results showed that before hypoxia (0 h), the Caco-2 cells were densely arranged, with ZO-1  and occlThe regulation of actin networks is critical to numerous physical cellular processes, including cell contraction, adhesion, migration, and division. Based on the abovementioned results, we then focused on F-actin in hypoxia-treated Caco-2 cells . As showIt has been recognized that cell shape and mechanical force generation, to a large degree, are regulated by the dynamic mechanical behaviors of a diverse assortment of actin networks and bundles . The ratIt is well known that jasplakinolide and LatA can both cause a deregulation of the F-actin/G-actin balance . After CMany studies have shown that cell permeability is associated with TER. We further investigated the effect of jasplakinolide or LatA on cell permeability. As shown in ZO-1 and occludin play important roles in cell tight junction. To clarify whether disruption of actin dynamics can induce a change of TJ proteins, we further detected the protein level of ZO-1 and occludin. The results showed that compared to that at 0 h, the relative expression levels of the ZO-1 and occludin proteins gradually decreased after treatment with jasplakinolide  or LatA via TJ proteins.Based on the above results of TJ protein level, we continued to observe the effect of jasplakinolide or LatA on the distribution of TJ proteins in Caco-2 cells. The results showed that after 24 h of treatment with jasplakinolide or LatA, the morphology of Caco-2 cells became very irregular, with the cell body significantly retracted and gaps forming between the cells. The distribution of ZO-1  and occlThe previous studies have shown that the dynamic balance between actin polymerization and depolymerization in cells is mainly regulated by the activity of the cofilin protein. Thus, we further detected the changes in the expression of p-cofilin and total cofilin in Caco-2 cells. As shown in Damage to the intestinal barrier function is an important cause of bacterial translocation, inflammatory responses, sepsis, multiple organ dysfunction, high metabolic responses, and even death after severe burn injury . Previouin vitro (To further elucidate the molecular mechanism underlying intestinal TJ barrier damage in the early stages of burn injury, this study investigated the effect of hypoxia on the barrier function of human intestinal epithelial cell monolayers. The TER can be used as a sensitive indicator to evaluate epithelial or endothelial barrier function in vitro . We estaOur previous study showed that changes in F-actin in the intestinal epithelial cytoskeleton might be associated with the occurrence of intestinal barrier damage in the early stages of burn injury . This stCofilin is an actin depolymerizing factor that can bind to actin in eukaryotic cells. It is currently the only known actin-binding protein that can depolymerize F-actin and plays a very important role in the switch between F-actin and G-actin in cells . To furtIn summary, severe burn injury causes intestinal epithelial cell barrier function damage. Our results suggest that the molecular mechanism might be that hypoxia activates cofilin and induces F-actin depolymerization and an imbalance between F-actin and G-actin, resulting in a reduction in the expression and a change in the distribution of TJ proteins.All datasets generated for this study are included in the article/supplementary material.HS drafted the manuscript. JZ, WH, and PW performed parts of the experiments. FW conceived the experiments and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.